Amino acid-trehalose composition

ABSTRACT

An amino acid-trehalose composition comprising an amino acid composition which comprises proline, alanine, glycine, valine, threonine, leucine, histidine, lysine, isoleucine, arginine, phenylalanine, and tyrosine; and trehalose. The amino acid-trehalose composition has effects of compensating the reduction of the blood level of amino acids associated with severe exercise, of improving the exercise, of reducing the degree of fatigue after exercise and of recovering from fatigue. In addition, the administration of the composition permits the inhibition of the consumption of amino acids associated with exercise and any induction of fatigue accompanied by exercise.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to an amino acid-trehalosecomposition developed on the basis of the knowledge obtained from astudy of a composition which comprises amino acids contained in thesaliva secreted by the larvae of a hornet (belonging to the genus Vespa)and more particularly to an amino acid-trehalose composition as well asa liquid complement containing the same, which show effects ofcompensating the reduction of the blood level of amino acids associatedwith severe exercise, of improving the exercise, of reducing the degreeof fatigue after exercise and of recovering from fatigue.

[0002] The inventors of this invention have made researches on thesaliva secreted by the larvae of various kinds of hornets and have madeit clear that the liquid nutrient of the hornet permits the control ofthe formation of substances which become a cause of the fatigue duringexercise, prevention of any reduction of the blood sugar level and theimprovement of the moving ability (see Japanese Patent No. 2,518,692).Moreover, it has been elucidated that the working mechanism of theliquid nutrient is to accelerate the use of fats as energy sources forexercise (Abe, et al., J. Physical Fitness & Sports Med., 1995, 44:225).There have also been suggested that Vespa amino acid mixture (VAAM)which is a main component of the liquid nutrient shows a variety ofeffects such as recovery from fatigue associated with exercise, inaddition to the aforementioned functions (see, for instance, JapaneseUn-Examined Patent Publication (hereunder referred to as “J.P. KOKAI”)Nos. Hei 4-95026 (Japanese Patent No. 2873493), Hei 4-112825 (JapanesePatent No. 2873497), Hei 6-336426 and Hei 6-336432).

[0003] On the other hand, it has been well-known that the amino acidbalance in the blood is put into disorder due to the fatigue associatedwith exercise (see T. Bazzarre et al., J. Am. Collage Nutr., 1992,11:531). It has been believed that the balance is destroyed since themuscular tissues are destroyed and/or worn due to the stress associatedwith exercise. Up to now, however, the physiological meaning andsignificance thereof have never attracted special interest.

[0004] The inventors have further investigated the amino acidconcentration in the blood observed after exercise and the amino acidcomposition of VAAM and as a result, have found that the amino acidcomposition of VAAM is correlated to the blood amino acids reduced dueto fatigue after exercise. In other words, it has been found that theamino acids severely reduced in a person fatigued from exercise arepresent in VAAM in a higher concentration. For this reason, it isbelieved that the supplementation of these amino acids consumed duringexercise is indispensable to the improvement of exercise and quickrecovery from fatigue (J.P. KOKAI No. Hei 9-249556).

[0005] On the other hand, it has been demonstrated that trehalose caninduce a considerable increase in the concentration of a non-esterifiedfatty acid (NEFA) in the mouse serum during exercise (J.P. KOKAI No. Hei5-186353). In this regard, the liquid nutrient of the larvae of a hornetincludes a considerable amount of trehalose (Abe, et al., Comp. Biochem.Physiol., 1991, 99C:79). Thus, it is expected that the simultaneousadministration of trehalose and VAAM permit higher improvement of theexercise performance.

SUMMARY OF THE INVENTION

[0006] Accordingly, it is an object of the present invention to providean amino acid-trehalose composition having effects of compensating thereduction of the blood level of amino acids associated with vigorousexercise, of improving the exercise performance, of relieving the degreeof fatigue after exercise and of recovering from fatigue and inparticular a liquid complement containing the same.

[0007] It is another object of the present invention to provide a methodfor controlling any change in the amino acid concentration in the blood,which is observed after vigorous exercise, and for ensuring a desiredhigh level thereof as well as an amino acid-trehalose composition, inparticular, a liquid complement containing the same.

[0008] According to an aspect of the present invention, there isprovided an amino acid-trehalose composition which comprises an aminoacid composition containing proline, alanine, glycine, valine,threonine, leucine, histidine, lysine, isoleucine, arginine,phenylalanine, and tyrosine; and trehalose.

[0009] In the specification and claims, the term ‘trehalose’ meansα,α-trehalose.

[0010] According to another aspect of the present invention, there isalso provided a liquid complement containing the foregoing aminoacid-trehalose composition.

[0011] According to a further aspect of the present invention, there isprovided a method for controlling any change in the amino acidconcentration of the blood associated with severe exercise, whichcomprises the step of administering an effective amount of the foregoingamino acid-trehalose composition to an animal, in particular a mammalincluding human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a bar graph showing the lactic acid level in the bloodobserved after administering a variety of liquid nutrients to mice andthen forcing the animal to swim.

[0013]FIG. 2 is a bar graph showing the blood sugar level observed afteradministering a variety of liquid nutrients to mice and then forcing theanimal to swim. The symbol “*” means that the corresponding value has asignificant difference (p<0.05) with respect to the groups to which VAAMis administered.

[0014]FIG. 3 is a bar graph showing the free fatty acid level in bloodobserved after administering a variety of liquid nutrients to mice andthen forcing the animal to swim. The symbol “*” means that thecorresponding value has a significant difference (p<0.05) with respectto the groups to which VAAM is administered.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] According to a preferred embodiment of the present invention, theamino acid-trehalose composition comprises at least one member selectedfrom the group consisting of aspartic acid, serine, glutamic acid andmethionine, in addition to the foregoing essential components. Accordingto a more preferred embodiment of the present invention, the aminoacid-trehalose composition further comprises at least one memberselected from the group consisting of taurine, β-alanine, γ-aminobutyricacid, ethanolamine, ammonia, ornithine, 1-methylhistidine, and3-methylhistidine.

[0016] In the amino acid-trehalose composition of the present invention,the weight ratio of the amino acid composition to the trehalosepreferably falls within the range: 0.45-1.6/0.5-5.0 and more preferably0.8-1.6/1.0-4.0.

[0017] The amino acid-trehalose composition of the present inventioncomprises an amino acid composition and trehalose, as has been discussedabove.

[0018] The amino acid composition used in the amino acid-trehalosecomposition of the invention is preferably those comprising 4 to 30moles of proline, 4 to 16 moles of alanine, 7 to 25 moles of glycine, 4to 16 moles of valine, 2 to 15 moles of threonine, 2 to 12 moles ofleucine, 1 to 12 moles of histidine, 5 to 12 moles of lysine, 3 to 9moles of isoleucine, 2 to 5 moles of arginine, 0.5 to 5 moles ofphenylalanine, and 1 to 9 moles of tyrosine.

[0019] The amino acid composition used in the amino acid-trehalosecomposition of the invention is more preferably those (VAAM analogue)comprising 4 to 30 moles of proline, 4 to 12 moles of alanine, 7 to 20moles of glycine, 4 to 8 moles of valine, 2 to 15 moles of threonine, 2to 12 moles of leucine, 1 to 5 moles of histidine, 5 to 11 moles oflysine, 3 to 9 moles of isoleucine, 2 to 5 moles of arginine, 0.5 to 5moles of phenylalanine, 1 to 9 moles of tyrosine, and 0.1 to 5 moles ofmethionine.

[0020] Preferably used herein further include the foregoing compositions(VAAM analogue) which further comprise not more than 1 mole of asparticacid, not more than 5 moles of serine and not more than 4 moles ofglutamic acid.

[0021] Preferably used herein also include the foregoing compositionswhich further comprise not more than 3 moles of taurine, not more thanone mole of β-alanine, not more than 0.5 mole of γ-aminobutyric acid,not more than 3 moles of ethanolamine, not more than 2 moles of ammonia,not more than 3 moles of ornithine, not more than one mole of1-methylhistidine, and not more than one mole of 3-methylhistidine.

[0022] The particularly preferred amino acid composition used in theamino acid-trehalose composition of the invention is those (HVAAManalogue) comprising 12.6 to 23.4 moles of proline, 8.4 to 15.6 moles ofalanine, 13.3 to 24.9 moles of glycine, 8.2 to 15.4 moles of valine, 5.0to 9.4 moles of threonine, 4.3 to 8.1 moles of leucine, 1.8 to 11.9moles of histidine, 1.7 to 3.3 moles of serine, 6.0 to 11.2 moles oflysine, 3.1 to 5.9 moles of isoleucine, 2.2 to 10.4 moles of glutamicacid, 2.4 to 4.6 moles of arginine, 2.6 to 5.0 moles of phenylalanine,and 4.2 to 7.8 moles of tyrosine.

[0023] More preferred HVAAM analogue has the following composition: 14.4to 21.6 moles of proline, 9.6 to 14.4 moles of alanine, 15.2 to 23.0moles of glycine, 9.4 to 14.2 moles of valine, 5.8 to 8.7 moles ofthreonine, 5.0 to 7.5 moles of leucine, 2.0 to 11.0 moles of histidine,2.0 to 3.0 moles of serine, 6.8 to 10.4 moles of lysine, 3.6 to 5.4moles of isoleucine, 2.5 to 9.6 moles of glutamic acid, 2.8 to 4.2 molesof arginine, 3.0 to 4.6 moles of phenylalanine, and 4.8 to 7.2 moles oftyrosine.

[0024] The composition of the particularly preferred HVAAM analogue isas follows: 16.2 to 19.8 moles of proline, 10.8 to 13.2 moles ofalanine, 17.1 to 21.1 moles of glycine, 10.6 to 13.0 moles of valine,6.4 to 8.0 moles of threonine, 5.5 to 6.8 moles of leucine, 2.3 to 10.1moles of histidine, 2.2 to 2.8 moles of serine, 7.7 to 9.5 moles oflysine, 4.0 to 5.0 moles of isoleucine, 2.8 to 8.8 moles of glutamicacid, 3.1 to 3.9 moles of arginine, 3.4 to 4.2 moles of phenylalanine,and 5.4 to 6.6 moles of tyrosine.

[0025] In the amino acid composition (HVAAM analogue) used in thepresent invention, the molar amount of histidine preferably ranges from6.4 to 11.9 moles, more preferably 7.2 to 11.0 moles and most preferably8.1 to 10.1 moles. In addition, the molar amount of glutamic acidpreferably ranges from 5.6 to 10.4 moles, more preferably 6.4 to 9.6moles and most preferably 7.2 to 8.8 moles.

[0026] The amino acid composition (HVAAM analogue) used in the presentinvention may comprise, in addition to the foregoing amino acids,methionine (in an amount preferably ranging from 0.3 to 0.7 mole % andmore preferably 0.4 to 0.6 mole %), aspartic acid (in an amountpreferably ranging from 0.1 to 0.3 mole %), taurine (Tau) (in an amountof preferably not more than 3 mole %), phospho-ethanolamine (P-EtAm) (inan amount of preferably not more than 2 mole %), cystine (Cys) (in anamount of preferably not more than 0.5 mole %), β-alanine (β-Ala) (in anamount of preferably not more than one mole %), γ-aminobutyric acid(GABA) (in an amount of preferably not more than 0.5 mole %), ornithine(Orn) or ethanolamine (EtAm) (in an amount of preferably not more than 3mole %), ammonia (NH₃) (in an amount of preferably not more than 2 mole%), 1-methylhistidine (1-MeHis) (in an amount of preferably not morethan 3 mole %), 3-methylhistidine (3-MeHis) (in an amount of preferablynot more than 1 mole %).

[0027] The amino acids present in the amino acid composition used in theinvention are particularly preferably L-amino acids.

[0028] The amino acid composition of the present invention may beprepared by mixing the foregoing amino acids commercially available in adesired mixing ratio specified above. Moreover, if the composition isused in the form of a liquid complement, it is sufficient to dissolvethe composition in water. In general, the composition is prepared in theform of a uniform powdery mixture and may be dissolved in water prior tothe practical use thereof. The temperature for preparing and storing thecomposition of the present invention is not restricted to any specificrange, but the composition is preferably prepared and stored at atemperature of not more than room temperature. The composition of theinvention has a slightly bitter taste, does not have any toxicity evenwhen administering it to a mouse at a level of 20 g/kg and the LD₅₀value thereof is much higher than 20 g/kg.

[0029] The composition of the present invention may effectively be usedin medicines and foods such as beverages. If it is used in the form of amedicine, the dosage form thereof is not restricted to any specific one.Thus, the composition may be used in any dosage form and administeredthrough the usual routes. For instance, it may be administered orally,through rectum, through injection and intravenous infusion. If it isorally administered, the composition may be used in the form of acomposition containing the ingredients defined above or in the form ofpharmaceutical preparations, which comprises the composition incombination with pharmaceutically acceptable carriers and/or vehicles,such as tablets, capsules, powders, troches and syrups. However, thecomposition is preferably in the dosage form of, for instance, a liquidpreparation and administered orally, since the absorption of thecomposition by the body may require a long period of time if it is usedin the form of a solid preparation such as a tablet or a powder. In thiscase, the composition is preferably administered in the form of anaqueous solution along with an appropriate additive, for instance, asalt such as sodium chloride, a buffering agent and/or a chelatingagent. If the composition is used as an injection, an appropriatebuffering agent, an isotonizing agent or the like may be added to thecomposition, then the resulting mixture is dissolved in sterilizeddistilled water to give an injectable solution and the solution may beintravenously administered through, for instance, instillation.

[0030] If it is used as a food, the composition to which a flavor isgiven may be formed into a drink such as a refreshing beverage or apowdered drink such as those prepared by drying the mixture through aspray-drying, freeze-drying or micro fine powder-forming method to givepowder and then encapsulating it in a capsule, or a tablet.

[0031] The composition of the present invention has quite low toxicityand therefore, the dose thereof may widely vary. The dose may dependupon the mode (route) of administration and the intended purposes of thecomposition, but the amount of the solid content thereof ranges from 1to 12 g/unit dose or 3 to 18 g/day, preferably 2 to 4 g/unit dose or 6to 12 g/day.

[0032] If the composition is used in the form of a liquid complementbefore, during and/or after exercise, the composition is used oradministered in the form of a solution having a concentration of 0.3 to3.8% by weight in an amount ranging from 200 to 500 ml/unit dose over 1to 3 times per day. The injectable solution may be a 0.8 to 3.8% byweight aqueous solution and may be administered in a unitary doseranging from 100 to 400 ml and preferably 150 to 300 ml.

[0033] The present invention will hereinafter be described in moredetail with reference to the following working Examples and TestExamples, but the present invention is not restricted to these specificExamples at all.

EXAMPLE 1

[0034] There were dissolved, in distilled water, amino acid components(total amount: 1.8 g) in the form of an amino acid composition (VAAManalogue) listed in the following Table 1 and 2.0 g of trehalose to givean aqueous solution (3.8 g/100 ml).

TEST EXAMPLE

[0035] This test was performed in order to demonstrate the effect of theamino acid-trehalose composition of the present invention, i.e., thereduction of exercise load during sustaining exercise and morespecifically, the test was an experiment carried out using mice to whichthe composition was administered prior to the initiation of theexercise.

[0036] This experiment was carried out according to the method disclosedin Jpn. J. Phys. Fitness Sports Med., 1995, 44:225-238. Morespecifically, mice (male; ddY) (6-week-old; 5 animals per group) werefasted at room temperature for 16 hours, followed by oral administrationof a 1.8% by weight aqueous solution containing the composition VAAM orHVAAM as set forth in Table 1 or trehalose; an aqueous solutioncontaining 1.8% by weight of VAAM and 2% by weight of trehalose; or anaqueous solution containing 1.8% by weight of VAAM analogue as set forthin Table 1 and 2% by weight of trehalose in an amount of 37.5 μl/g bodyweight and thereafter giving the mice a rest at room temperature for 30minutes. Then the mice were forced to swim in a running water pool (awater tank having a diameter of 32 cm and a depth of 30 cm, which wasfilled with water maintained at 35° C. and in which the water wascirculated at a rate of 8 m/min using a circulator) for 30 minutes.After forcing the mice to swim, they were inspected for the lactic acidlevel in the blood, the blood sugar (glucose) level and the free fattyacid level in the blood.

[0037] The lactic acid level in the blood was determined using aclinical reagent available from Sigma Company, by measuring absorbanceat 340 nm due to NADH, each molecule of which was formed when lacticacid was converted into pyruvic acid because of the action of lacticacid dehydrogenase. The free fatty acid level in the serum wasdetermined by centrifuging the collected blood and then quantitativelyanalyzing the fatty acids present in the supernatant. More specifically,the hydrogen peroxide formed by the action of acyl-CoA synthetic enzymeand acyl-CoA oxidase was reacted with a peroxidase, followed bydetermination of the absorbance at 550 nm due to the stained3-methyl-N-ethyl-N-(2-hydroxyethyl)aniline and 4-aminoantipyrine to thusdetermine the free fatty acid level.

[0038] The results thus obtained are summarized in the following Table 2and the attached FIGS. 1, 2 and 3. TABLE 1 Molar Ratio of Amino AcidKind of VAAM Amino Acid analogue VAAM HVAAM Pro 18.40 18.00 18.00 Ala6.14 6.00 12.00 Gly 19.53 19.10 19.10 Val 6.03 5.90 11.80 Thr 7.36 7.207.20 Leu 6.34 6.20 6.20 His 2.66 2.60 2.60 Ser 2.56 2.50 2.50 Lys 8.798.60 8.60 Ile 4.60 4.50 4.50 Glu 3.27 3.20 3.20 Arg 3.58 3.50 3.50 Met0.51 0.50 0.50 Asp 0.21 0.20 0.20 Phe 3.89 3.80 3.80 Tyr 6.13 6.00 6.00Trp 0.00 2.20 2.20

[0039] TABLE 2 VAAM analogue + VAAM + VAAM Tre Tre Tre HVAAM Lactic Acid46.72 ± 46.34 ± 46.27 ± 46.69 ± 45.74 ± Level (mg/dl)  4.06  2.75  2.72 1.19  3.87 Blood Sugar 74.34 ± 86.27 ± 86.59 ± 83.14 ± 94.89 ± Level(mg/dl)  3.67  2.46  2.49  3.45  3.65 NEFA (mEq/l)  0.58 ±  0.70 ±  0.72±  0.97 ±  0.87 ±  0.01  0.05  0.05  0.06  0.04

[0040] The groups of mice to which HVAAM, trehalose (Tre),VAAM+trehalose and VAAM analogue+trehalose were administered,respectively did not show any significant difference in the lactic acidlevel in the blood observed after applying thereto the load of swimmingover 30 minutes, as compared with that observed for the group to whichVAAM was administered (see FIG. 1).

[0041] On the other hand, the groups of mice to which 1.8% VAAManalogue+2% trehalose, 1.8% VAAM+2% trehalose and 1.8% HVAAM wereadministered, respectively showed the blood sugar levels significantlyhigher than that observed for the group to which 1.8% VAAM wasadministered (see FIG. 2). Moreover, the groups to which 1.8% VAAManalogue+2% trehalose, 1.8% VAAM+2% trehalose; 2% trehalose; and 1.8%HVAAM were administered, respectively also showed the free fatty acidlevels in the blood significantly higher than that observed for thegroup to which 1.8% VAAM was administered (see FIG. 3).

[0042] The foregoing results clearly indicate that the mixture oftrehalose with VAAM analogue, VAAM or HVAAM not only suppresses theincrease of the lactic acid level in the blood during exercise to anextent almost comparable to that observed for VAAM, but alsosignificantly suppresses the reduction of the blood sugar level duringexercise as compared with those observed for VAAM or trehalose, and havean effect of significantly increasing the free fatty acid level in theblood. Accordingly, it is clear that the mixture of trehalose with VAAManalogue, VAAM or HVAAM has an effect of improving the functionssuperior to the moving ability-improving effect of VAAM.

[0043] As has been described above in detail, the composition of thepresent invention has effects of compensating the reduction of the bloodlevel of amino acids associated with severe exercise, of improving theexercise, of reducing the degree of fatigue after exercise and ofrecovering from fatigue. In addition, the administration of thecomposition of the present invention permits the inhibition of theconsumption of amino acids associated with exercise and any induction offatigue accompanied by exercise.

What is claimed:
 1. An amino acid-trehalose composition comprising anamino acid composition which comprises proline, alanine, glycine,valine, threonine, leucine, histidine, lysine, isoleucine, arginine,phenylalanine, and tyrosine; and trehalose, wherein the amino acidcomposition comprises 4 to 30 moles of proline, 4 to 16 moles ofalanine, 7 to 25 moles of glycine, 4 to 16 moles of valine, 2 to 15moles of threonine, 2 to 12 moles of leucine, 1 to 12 moles ofhistidine, 5 to 12 moles of lysine, 3 to 9 moles of isoleucine, 2 to 5moles of arginine, 0.5 to 5 moles of phenylalanine, and 1 to 9 moles oftyrosine.
 2. The amino acid-trehalose composition of claim 1 furthercomprising at least one member selected from the group consisting ofaspartic acid, serine, glutamic acid and methionine.
 3. The aminoacid-trehalose composition of claim 2 further comprising at least onemember selected from the group consisting of taurine, β-alanine,γ-aminobutyric acid, ethanolamine, ammonia, ornithine,1-methylhistidine, and 3-methylhistidine.
 4. The amino acid-trehalosecomposition of claim 1 wherein the weight ratio of the amino acidcomposition to trehalose falls within the range: 0.45-1.6/0.5-5.0. 5.The amino acid-trehalose composition of claim 1 wherein the amino acidcomposition comprises 4 to 30 moles of proline, 4 to 12 moles ofalanine, 7 to 20 moles of glycine, 4 to 8 moles of valine, 2 to 15 molesof threonine, 2 to 12 moles of leucine, 1 to 5 moles of histidine, 5 to11 moles of lysine, 3 to 9 moles of isoleucine, 2 to 5 moles ofarginine, 0.5 to 5 moles of phenylalanine, 1 to 9 moles of tyrosine and0.1 to 5 moles of methionine.
 6. The amino acid-trehalose composition ofclaim 5 wherein the amino acid composition further comprises not morethan 1 mole of aspartic acid, not more than 5 moles of serine and notmore than 4 moles of glutamic acid.
 7. The amino acid-trehalosecomposition of claim 6 wherein the amino acid composition furthercomprises not more than 3 moles of taurine, not more than 1 mole ofβ-alanine, not more than 0.5 mole of γ-aminobutyric acid, not more than3 moles of ethanolamine, not more than 2 moles of ammonia, not more than3 moles of ornithine, not more than 1 mole of 1-methylhistidine, and notmore than 1 mole of 3-methylhistidine.
 8. The amino acid-trehalosecomposition of claim 1 which is in the form of a transfusion.
 9. Theamino acid-trehalose composition of claim 8 wherein the aminoacid-trehalose composition further comprises at least one memberselected from the group consisting of aspartic acid, serine, glutamicacid and methionine.
 10. The amino acid-trehalose composition of claim 9wherein the amino acid-trehalose composition further comprises at leastone member selected from the group consisting of taurine, β-alanine,γ-aminobutyric acid, ethanolamine, ammonia, ornithine,1-methylhistidine, and 3-methylhistidine.
 11. A method for compensatingfor the reduction of amino acid blood levels associated with vigorousexercise, comprising administering to an animal in need thereof aneffective amount of the amino acid-trehalose composition of claim 1 tocompensate for the reduction of amino acid blood levels.
 12. The methodof claim 11 wherein the animal is a mammal.
 13. The method of claim 12wherein the mammal is human.
 14. A food comprising the aminoacid-trehalose composition of claim 1 and a physiologically acceptablecarrier.
 15. The food of claim 14 wherein the amino acid-trehalosecomposition further comprises at least one member selected from thegroup consisting of aspartic acid, serine, glutamic acid and methionine.16. The food of claim 15 wherein the amino acid-trehalose compositionfurther comprises at least one member selected from the group consistingof taurine, β-alanine, γ-aminobutyric acid, ethanolamine, ammonia,ornithine, 1-methylhistidine, and 3-methylhistidine.
 17. The food ofclaim 14 which is in the form of a beverage, a powdered drink or atablet.